Preparation for deactivating viruses and process for producing same

ABSTRACT

The invention relates to a process and a preparation for deactivating viruses inside living human and animal organisms by application of a terpene obtainable from aromatic plants by steam distillation. The terpenes cited are: black pepper oil, cinnamon flower oil, cardamon oil, linallyl acetate, cinnamic aldehyde, safrol, carvon and cis/trans citral.

This application is a division, of application Ser. No. 184,135, filedSept. 4, 1980 now U.S. Pat. No. 4,402,950 which is a continuation ofSer. No. 005,764, filed Jan. 23, 1979, now abandoned.

The invention relates to a process and a preparation for deactivatingviruses inside living human and animal organisms. During use thereof,cell damage and other harmful side effects in the organisms should beprevented.

The process according to the invention is characterised by the use of aterpene obtainable by steam distillation from aromatic plants, in adaily dose of 5 to 500 mg (mmiligrams), preferably 25 to 100 mg per 50kg (kilograms) of the weight of the living organism.

These terpenes demonstrate a viricidal activity (i.e. a damaging effecton viruses) in a concentration which is one or more powers of ten lowerthan the concentration at which these terpenes have toxic effects onliving cells. This wide range gives a degree of play in tolerance whichis advantageous from the dosage point of view and thus makes it possibleto administer these terpenes safely in veterinary and human medicine.

Since these terpenes can be obtained from aromatic plants which havebeen used for feeding animals and humans for many years and have provedharmless in the doses in question, it is also to be expected that thequantities of terpenes to be used according to the invention will notcause any serious, harmful side effects.

Terpenes or mixtures of terpenes which have proved suitable are thoseconsisting of black pepper oil, cinnamon flower oil, cardamom oil,linallyl acetate, cinnamic aldehyde, safrol, carvon and cis/transcitral, used individually or mixed together.

A pharmaceutical preparation according to the invention, i.e. fordeactivating viruses inside living human and animal organisms, isprepared by obtaining one or more of these terpenes by steamdistillation from the parts of the aromatic plants where the relevantterpenes are contained and then mixing them into a pharmaceuticalcarrier substance in a ratio of 1:100 to 20:100.

A pharmaceutical preparation according to the invention, i.e. fordeactivating viruses inside living human and animal organisms, consistsof one or more of the terpenes listed, mixed into a pharmaceuticalcarrier substance in a ratio of 1:100 to 20:100.

The terpenes used can be obtained from aromatic plants by steamdistillation as follows:

black pepper oil from the peppercorns of Piper nigrum;

cinnamon flower oil from the flowers of Cinnamonum Cassia;

cardamom oil from the seeds of Elctiaria Cardamomum;

linallyl acetate from the flowers of Lavandula;

cinnamic aldehyde from the bark of Cinnamomum ccylanicum;

safrol from the roof of Sassafras;

carvon from the fruit of Carum carvi, and

cis/trans citral from the leaves of Cymbopogon citratus.

Instead of these natural terpenes, identical synthetic terpenes may beused, if available. However, natural terpenes obtained from aromaticplants are preferred.

The activity obtained according to the invention is demonstrated bycomparison tests as follows.

Cell cultures were cultivated in various culture vessels under optimalculture conditions from permanent strains of the types "Girardi Heart"(GH), "Flow 12000" (FL), "Intestine 407" (IX) and "Vero Kidney" (VE), sothat a layer of cell culture containing about 0.25 mg of cell substanceformed on the bottom of the vessels.

A suspension of virus particles of the Adeno Type 6 virus was alsoadded.

For the total of eight terpenes listed in Table I, twenty cell culturesof each type of cell were prepared. The twenty cell cultures of eachtype of cell were treated with differing amounts of the relevant terpenein the following manner.

The first two cell cultures were given 10⁵ mg of terpene per 10 kg ofcell substance. The next two cell cultures were given 10⁴ mg of terpenebased on 10 kg of cell substances. The next two cell cultures were given10³ mg of terpene based on 10 kg of cell substance and so on to the lasttwo of these twenty cell cultures, which were given 0.1 mg of terpeneper 10 kg of cell substance. Thus, in each case, two similar cellcultures were treated with the same amount of the same terpene. Forcontrol purposes, one of these two identical cell cultures was left asit was, whilst the other was inoculated with 5×10⁶ virus particles per0.25 mg of cell substance, in addition to the virus suspension used. Thesame procedure was also followed with the other cell cultures andterpenes.

The cell cultures thus treated were left to stand and observed afterfour days and six days. This observation was carried out by microscopicinvestigation of the cell culture for cell damage. The damage observedwas divided into four stages, as follows:

    ______________________________________                                        State 0                                                                             means    no damage                                                      Stage 1                                                                             means    slackened growth of the cell formations                        Stage 2                                                                             means    the cells become spherical and detach them-                                   selves from the bottom                                         Stage 3                                                                             means    the cell structures are substantically or                                     totally destroyed.                                             ______________________________________                                    

It was found that the inoculated cell cultures which were protected witha very small amount of terpene reached stage 3 or 2, as the viruses haddamaged the cells. The inoculated cell cultures containing a very largeamount of terpene also reached stage 3 or 2, as the cells were damagedby the excessive terpene. However, the inoculated cell culturescontaining only a moderate amount of terpene were at stage 0, i.eundamaged. Thus, the moderate amount of terpene damaged the virusessufficiently and protected the cells from viral attack, without thecells being damaged directly by the terpene. The terpene concentrationswith which stage 0 and, in some isolated cases, stage 1 were observed inthe inoculated cell cultures after four and six days result insufficient damage to the viruses without damaging the cells, and aregiven in Table I.

Column 1 of Table I gives the terpene used, the second column gives thetreated cell strain, abbreviated as hereinbefore, and the third columngives the amount of terpenes used in mg, based on 10 kg of treated cellsubstance, for the range of concentrations in which no appreciable celldamage (i.e. stage 0) was observed. This range is the therapeutic rangewhich in each case extends over several powers of ten. Thus, for all theterpenes listed in the Table, the desired viricidal activity occurs at aconcentration which is several powers of ten lower than the lowestconcentration at which cell damage was observed, i.e. at which themicroorganisms to be protected could be damaged.

                  TABLE I                                                         ______________________________________                                                               range of viricidal con-                                                       centration at which no cell                                                   damage was observed, in mg                                          treated cell                                                                            of terpene per 10 kg of                                Terpene      substance treated cell substance                                 ______________________________________                                        black pepper oil                                                                           GH        10.sup.3   to 0.1                                      ( Oleum Piperis nigri)                                                                     FL        100        to 0.1                                                   IN        100        to 1                                                     VE        100        to 0.1                                      Cinnamon flower oil                                                                        GH        10.sup.3   to 0.1                                      ( Oleum Cassiac flores)                                                                    FL        10.sup.3   to 0.1                                                   IN        100        to 0.1                                                   VE        100        to 0.1                                      Cardamon oil GH        100        to 1                                        ( Oleum Cardamoni)                                                                         FL        100        to 1                                                     IN        100        to 1                                                     VE        100        to 10                                       Linallyl acetate                                                                           GH        100        to 0.1                                                   FL        100        to 1                                                     IN        100        to 1                                                     VE        100        to 1                                        cinnamic aldehyde                                                                          GH        100        to 1                                                     FL        100        to 1                                                     IN        100        to 1                                                     VE        100        to 1                                        sairol       GH        100        to 1                                                     FL        100        to 1                                                     IN        100        to 10                                                    VE        100        to 1                                        carvoa       GH        100        to 1                                                     FL        100        to 1                                                     IN        100        to 1                                                     VE        100        to 1                                        cis/trans citral                                                                           GH        10         to 1                                                     FL        10         to 1                                                     IN        100        to 1                                                     VE        100        to 1                                        ______________________________________                                    

EXAMPLE 1 (Injection solution)

50 g of black pepper oil are dissolved in 2 l (liters) of1,2-propanediol. The solution is sterilized in the autoclave for 50minutes at 121° C. (Celsius), then cooled and poured into ampoules in 2g amounts.

An ampoule contains 50 mg of black pepper oil and contains an averagedaily dose for an adult weighing 70 kg, for the therapy and proplylaxisof influenza infections. For human and animal patients of other weights,the daily dose must be modified accordingly in proportion to thepatient's weight.

The mixing ratio of terpene to 1,2-propanediol in this example is2.5:100; other mixing ratios for injection solutions are possible,within the range 1:100 to 5:100, but the daily dose of the injectionsolution must then be adjusted to the different terpene content of theinjection solution.

EXAMPLE 2 (Aerosol)

325 g of black pepper oil are dissolved in 631.8 g of other mixed with1,305.07 g of ethanol. To this solution are added 31.6 g of ester ofcastor oil fatty acids with ethoxylated glycerol and 210.6 g ofcapryl/capric acid triglyceride. 2.68 g of this mixture, together with2.527 g of difluorodichloromethane as the propellant, are packed in aspray can with a capacity of 20 cc (cubic centimeters). The spray can issealed and comprises a metering valve which releases a specific amountof mixture each time it is actuated, and this mixture is then vaporisedas an aerosol under the pressure of the difluorodichloromethane.

Corresponding adjustment and dimensions of the metering valve ensurethat, on each application, a single dose containing 6.5 mg of blackpepper oil is released.

For the treatment and prevention of influenza infections, the aerosol issprayed into the mouth or nose and inhaled. A suitable treatment for anadult weighing 70 kg is eight such single doses per day, containing atotal of 8×6.5=50 mg of black pepper oil.

The aerosol can also be used to treat areas of the skin affected byvirus infections, in which case an area of 50 cm² (square centimeters)is sprayed with seven spray doses each containing 6.5 mg of black pepperoil.

The mixing ratio of terpene to the aerosol substance is 12:100 in thisexample; other mixing ratios for the aerosol are possible, in the rangefrom 5:100 to 20:100, but then the daily dose must be adjusted to themodified terpene content of each spray portion.

EXAMPLE 3 (Capsules)

A capsule filling is prepared from a mixture of 12.5 g of black pepperoil and 12.5 g of cinnamon flower oil and 3 g of soya lecithin as theemulsifier. Each capsule contains 28 mg of this filling and is sealedwith a capsule shell consisting of 87.5 mg of gelatine and 37.5 mg ofglycerol.

For the treatment and prevention of influenza infections, one to fourcapsules per day are administered orally to an adult patient weighing 70kg; if more than one capsule is taken, they are spread out over the day.

One capsule contains 25 mg of terpene; however, variations are possible,with a capsule containing from 10 to 50 mg of terpene, but then thedaily dose must be adjusted to the modified terpene content.

EXAMPLE 4 (Stick)

1 g of black pepper oil is mixed into a stable carrier composition. Thecarrier composition consists of 59.84 g of Vaseline album and 39.16 g ofparaffin and is thoroughly mixed with the terpene at 70° C. and thenpoured into a mould to form a stick and hardened by cooling.

For local use, the stick is rubbed on to the skin and distributed sothat 1 ml (milliliter) of stick compound-which contains 5 mg of terpenein this example-is distributed over 50 cm² of skin. This can be repeated3 times daily.

The mixing ratio of terpene to carrier composition is 1:100 in thisexample; other mixing ratios are possible, in the range from 1:100 to5:100.

EXAMPLE 5 (Ointment)

3.2 g of Paraffinum durum and 86.8 g of white Vaseline are heated to 60°C. and stirred. 10 g of cinnamon flower oil are mixed into the warmmixture. The mixture is cooled and can be used as an ointment for localapplication. About 0.1 ml of ointment-containing about 5 mg ofterpene-are spread over 50 cm² of skin. This can be repeated 8 times aday.

The mixing ratio of terpene to the mixture of Paraffinum durum and whiteVaseline is 11:100 in this example, but other mixing ratios for theointment are also possible, within the range from 5:100 to 20:100.

EXAMPLE 6 (Plaster)

A vapour-proof plaster film is produced from textile material madevapour-proof by coating with plastics on the underside. On the otherside (the contact side) the plaster is coated with a plaster compound ina layer 1 mm (millimeter) thick. To prepare the plaster compound, 97 gof lead plaster, 8 g of yellow wax, 9 g of dammar, 10 g of colophony and1 g of turpentine are mixed together, heated to 100° C. and stirreduntil the molten compound is no longer foaming. Then 5 g of black pepperoil are mixed in and the plaster compound is then applied to the contactside of the plaster film and hardened by cooling.

The plaster is placed with its contact side next to the skin and leftfor four hours. It can then be replaced by a new plaster.

The mixing ratio of terpene to plaster compound is 4:100 in thisexample; however, other mixing ratios are also possible within the rangefrom 1:100 to 10:100.

Using the preparations and treatments given in the examples, viralattacks can be prevented or stopped without causing any cell damage inthe treated organism or any other serious side effects.

The examples given are open to modification by using, instead of theterpene mentioned, the some amount of another terpene from Table I or amixture of several of these terpenes. In all these cases, the viricidalactivity can be obtained without having to take into account anyundesirable side effects.

We claim:
 1. A process for preparing a pharmaceutical composition fordeactivating viruses inside living human and animal organisms,comprising the steps ofderiving a terpene from black pepper oil by steamdistillation; and admixing said derived terpene in a pharmaceuticalcarrier in a mixing ratio of terpene to carrier of from about 1:100 toabout 20:100.
 2. The process of claim 1, wherein said plant is Pipernigrum.
 3. The process of claim 1, wherein said pharmaceutical carrieris 1,2-propanediol.
 4. A pharmaceutical aerosol composition for humansand animals for deactivating viruses, includinga terpene derived fromblack pepper oil; and an aerosol carrier vehicle in a ratio of betweenabout 5:100 and about 20:100 of terpene to carrier vehicle.
 5. Thecomposition of claim 4, wherein said mixing ratio of terpene to carriervehicle is about 12:100.
 6. The composition of claim 4, wherein saidcarrier vehicle includes ether, ethanol, an ester of a castor oil fattyacid with ethoxylated glycerol, capryl/capric acid triglyceride anddifluorodichloromethane.
 7. The composition of claim 6, wherein saidcarrier vehicle includes about 1 part by weight of ether, between about2 and about 5 parts by weight of ethanol, between about 0.02 and about0.1 parts by weight of an ester of a castor oil fatty acid withethoxylated glycerol, between about 0.2 and about 1 part by weight ofcapryl/capric acid triglyceride and between about 2 and about 6 parts ofdifluorodichloromethane.
 8. The composition of claim 4, wherein theratio of said black pepper oil to the aerosol carrier vehicle is about12:100.
 9. A pharmaceutical capsule composition for ingestion by humansand animals for deactivating viruses, including a terpene derived fromblack pepper oil; an emulsifier; and a capsule casing, the weight ofsaid terpene being between about 10 mg and about 50 mg and the weight ofsaid capsule being between about 80 mg and 200 mg.
 10. The compositionof claim 9, wherein said emmulsifier is soya lecithin.
 11. Thecomposition of claim 9, wherein said capsule casing consists of gelatineand glycerol in a ratio of between about 2:1 and about 3:1 by weight.12. The composition of claim 9, wherein the weight of said terpene isbetween about 10 mg and about 50 mg and the weight of said capsule isbetween about 80 and about 200 mg.
 13. A stable, spreadablepharmaceutical stick composition for external application by humans andanimals for deactivating viruses, including a terpene derived from blackpepper oil; and a carrier consisting of vaseline album and paraffin in aratio of between about 1:1 and about 2:1 by weight, the weight ratio ofterpene to carrier being between about 1:100 and about 5:100.
 14. Thecomposition of claim 13, wherein said weight ratio is about 1:100.
 15. Apharmaceutical ointment composition for external application by humansand animals for deactivating viruses, including a terpene derived fromblack pepper oil; and an ointment carrier consisting of Paraffinum durumand white vaseline in a weight ratio of between about 1:20 and about1:35, the weight ratio of terpene to carrier being between about 5:100and about 20:100.
 16. The composition of claim 5, wherein the ratio isabout 11:100.
 17. A pharmaceutical capsule composition for ingestion byhumans and animals for deactivating viruses, including a terpene derivedfrom black pepper oil; an emulsifier; and a capsule casing, the weightof said emulsifier being between about 1 mg and about 8 mg and theweight of said capsule being between about 80 mg and about 200 mg. 18.The composition of claim 17, wherein said emulsifier is soya lecithin.19. The composition of claim 17, wherein said capsule casing consists ofgelatine and glycerol in a ratio of between about 2:1 and about 3:1 byweight.